Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.

Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function.

The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.

Bradykinin and alpha-thrombin increase human umbilical vein endothelial macromolecular permeability by different mechanisms.

Bradykinin and alpha-thrombin both increase endothelial macromolecular permeability, however the mechanism for this effect is unclear. Human umbilical vein endothelial cell (HUVEC) permeability to human serum albumin was increased by 1 microM alpha-thrombin (AT) or bradykinin (BK), but the kinetics of the permeability response were different. Intracellular calcium mobilization of HUVEC by AT was increased, yet BK had no effect on intracellular calcium. Distribution of F-actin and content was increased by AT as early as 10 minutes after administration, yet BK had no affect on F-actin when compared to control. We hypothesized that BK may increase HUVEC permeability by producing matrix metalloproteinase-2 (MMP-2). The AT-treated HUVEC produced an intermediate 64 kDa MMP-2, whereas BK-treated HUVEC increased the intermediate 64 kDa MMP-2 and also an active 62 kDa MMP-2. Pre-treatment of the HUVEC with tissue inhibitor of matrix metalloproteinase-2 slightly decreased the AT-induced increase in macromolecular permeability and completely inhibited the BK-induced increase in macromolecular permeability.

Differential effects of ozone on lung epithelial lining fluid volume and protein content.

Urea dilution has been used to estimate the volume of epithelial lining fluid (ELF) in the respiratory tract. However, ELF volume may be overestimated as the result of rapid net diffusion of urea from tissues into the bronchoalveolar lavage (BAL) fluid. This study established a protocol for rat BAL in a manner that minimizes this problem and then used this procedure to examine the edemagenic effects of ozone (O3) exposure on ELF volume and the concentrations of ELF protein and albumin. One passage lavage with variable dwell times up to 30 s showed no difference in recovered urea, protein, and albumin and ELF volume between 0 and 4 s, but a progressive increase of each thereafter. The calculated concentrations of protein and albumin in ELF did not vary significantly with dwell time. By increasing the number of lavage passages from one to three, the amounts of recovered urea, protein, and albumin and estimated ELF volume were increased with each passage. Again, the calculated concentrations of protein and albumin in ELF did not vary appreciably. When a single lavage passage and no added dwell time were used, it was observed that exposure of rats to 2 but not 0.5 and 1 ppm O3 increased urea, protein, and albumin in the BAL immediately after 6 h exposure. In addition, at 18 h postexposure to 1 ppm O3, ELF volume increased only 21%, but protein and albumin concentrations in ELF were 2.3- and 4.5-fold of control values, respectively. A higher O3 concentration (2 ppm) moderately increased ELF volume (+83%) and exerted even greater effects on concentrations of ELF protein (7.8-fold) and albumin (19-fold) while lower O3 dosage (0.5 ppm) had no significant effect. SDS-PAGE analysis showed that small serum proteins including albumin were greatly enriched in lung BAL fluid of 1 ppm O3-exposed rats. These results demonstrate that movement of water and protein into the airspaces after O3 exposure is not strictly coupled, and that protein recovery by BAL should cautiously be used to indicate airspace edema as a result of O3 injury.

Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C.

We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the initial phase of the response, ET stimulated rapid formation of diacylglycerol due to rapid and transient activation of phosphatidyl inositol-specific phospholipase C and to rapid and prolonged activation of phospholipase D. Concurrently, ET stimulated translocation of PKC activity that reached a peak at 1 min and remained elevated for at least 20 min. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulation with ET inhibited total inositol phosphate formation by > 50%. Because each inositol phosphate isomer was equally affected, the target appears to be either phospholipase C or some upstream component of the receptor coupling mechanism. Activation of PKC was important for sustained response to ET. Treatment of cells with staurosporine significantly reduced sustained elevation of cytosolic free Ca2+ concentration ([Ca2+]i) normally seen with ET. We had previously shown that sustained elevation of [Ca2+]i initiated by ET was due to continued activity of L-type Ca2+ channels. Our current data suggest that PKC is important in this response. For example, staurosporine inhibited both ET-induced 45Ca2+ and Mn2+ entry occurring 10 min after stimulation of influx mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the sustained activity of Ca2+ entry mechanisms stimulated by ET. Finally, PMA initiated a slowly developing, sustained elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of temperature on bradykinin-induced arachidonate release and calcium mobilization in vascular endothelial cells.

The effect of decreased temperature on Ca(2+)-dependent arachidonic acid release was studied in vascular endothelial cells by investigating bradykinin (BK)-stimulated Ca2+ mobilization, inositol phosphate formation and arachidonic acid release. At both 37 degrees C and 22 degrees C, BK efficiently increased cytosolic Ca2+ concn. ([Ca2+]i). At 22 degrees C, peak [Ca2+]i was higher and returned to basal levels more slowly. Although this response was preceded by rapid formation of Ins(1,4,5)P3, the activity of phospholipase C was significantly impaired at 22 degrees C. To determine if Ins(1,4,5)P3 effectively mobilized intracellular Ca2+, we used saponin-permeabilized cells. Ins(1,4,5)P3, mobilized sequestered Ca2+ to a similar degree at 37 degrees C and 22 degrees C, although Ca2+ release was prolonged at 22 degrees C. In intact cells, BK mobilized intracellular Ca2+ stores and activated Ca2+ entry. The rate of 45Ca2+ entry was approx. 2-fold slower at 22 degrees C, even though the peak and duration of the rise in [Ca2+]i were higher and sustained at the lower temperature. TG mobilized intracellular Ca2+, activated Ca2+ entry and elevated [Ca2+]i at both temperatures. As with BK, the peak [Ca2+]i reached after thapsigargin treatment was higher at 22 degrees C. This effect of lower temperature on [Ca2+]i was most probably due to decreased Ca2+ efflux after a decrease in activity of the Ca(2+)-ATPase on the plasma membrane. Both A23187 and BK were shown to stimulate phospholipase A2 and arachidonic acid release at 22 degrees C. In each case, the rate and extent of release were decreased compared with that at 37 degrees C. Among several effects, lowering the temperature decreases the activity of phospholipase C, Ca(2+)-ATPase(s), Ca(2+)-entry mechanisms and phospholipase A2. Together, these effects lead to a higher and more prolonged elevation of [Ca2+]i, but a decrease in arachidonate release in response to BK.

Nitrogen dioxide exposure and urinary excretion of hydroxyproline and desmosine.

The relationship between average and peak personal exposure to nitrogen dioxide and urinary excretion of hydroxyproline and desmosine was investigated in a population of preschool children and their mothers. Weekly average personal nitrogen dioxide exposures for subjects who resided in homes with one or more potential nitrogen dioxide source (e.g., a kerosene space heater, gas stove, or tobacco smoke) ranged between 16.3 and 50.6 ppb (30.6 and 95.1 micrograms/m3) for children and between 16.9 and 44.1 ppb (12.8 and 82.9 micrograms/m3) for mothers. In these individuals, the hydroxyproline-to-creatinine and desmosine-to-creatinine ratios were unrelated to personal nitrogen dioxide exposure--even though continuous monitoring documented home nitrogen dioxide concentration peaks of 100-475 ppb lasting up to 100 h in duration. Significantly higher hydroxyproline-to-creatinine and desmosine-to-creatinine ratios were observed in children, compared with mothers (p < .001 and .003, respectively).

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways.

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

Respiratory allergy and the relationship between early childhood lower respiratory illness and subsequent lung function.

In a study of 159 school-age children whose histories of outpatient visits for lower respiratory illness (LRI) had been documented from early infancy, we observed lower mean levels of small airway function in boys who had experienced two or more episodes of wheezing-associated LRI before 6 yr of age. To determine whether allergy was an important factor influencing this result, we examined relationships among the results of RAST tests for seven common inhalant allergens and concurrent lung function in 126 subjects who consented to venipuncture. Increasing values for the sum of scores for the seven RAST tests were associated with progressively lower mean levels of small airways function in boys with histories of recurrent wheezing LRI during the preschool years. The association of allergy with lower levels of lung function was largely accounted for by dust mite allergy. RAST results were not correlated with lung function in boys who had experienced zero or 1 wheezing LRI before 6 yr of age or in girls. A history of recurrent wheezing LRI during the preschool years was also associated with significantly lower mean levels of small airways function in boys who had negative RAST tests. A subset of 49 boys was reevaluated after an average interval of 4 yr with RAST tests, spirometry, and methacholine challenge. Dust mite allergy was associated with an increased prevalence of bronchial hyperreactivity independent of early childhood wheezing LRI history.(ABSTRACT TRUNCATED AT 250 WORDS)

Home air nicotine levels and urinary cotinine excretion in preschool children.

We examined the extent of correlation between home air nicotine levels and urine cotinine/creatinine ratios (CCR) in 27 children who attended a research day care program where they were not exposed to environmental tobacco smoke (ETS) during the daytime hours. Average concentrations of nicotine in home air were determined by active air sampling during the evening and night hours on 2 consecutive days. Urine samples for cotinine and creatinine determinations were collected before, during, and after the two sampling periods. In addition, four sequential weekly urine samples for CCR were obtained from study children to determine the extent to which single determinations of CCR were representative for individual children. Fifteen children resided in homes with smokers, and 12 did not. Urine CCR consistently distinguished most exposed and unexposed children. However, three exposed children had urine CCRs that clustered routinely around the criterion CCR (30 ng/mg cotinine-creatinine) that best distinguished exposed and unexposed children. In children exposed to ETS in the home, there was a significant correlation between average home air nicotine levels and the average logarithm of urine CCR the two mornings after the home air monitoring periods (r = 0.68; p = 0.006). In study children, urine CCRs were remarkably stable over the 1-month observation period. Rank correlation coefficients for sequential weekly determinations of CCR were consistently greater than r = 0.88; p less than 0.0001.